Adeno-associated viruses (AAVs) are linear, single-stranded DNA viruses that belong to the Parvovirus family. The AAVs are infectious to cells of a wide range of species including human, and also infect non-dividing cells in which differentiation is terminated such as blood cells, muscles, or neural cells. Wild-type AAVs are not pathogenic to human. Also, the AAV particles are very stable physicochemically. From these features, the development of AAVs as vectors for gene transduction has been progressed.
The AAV particles comprise protein capsid having three capsid proteins (VP1, VP2, and VP3) and a single-stranded DNA genome surrounded therewith. The genome of the wild-type AAVs has a nucleotide sequence forming a T-shaped hairpin structure called ITR (Inverted Terminal Repeat) at both terminals, and a half of the linear single-stranded genome between the terminals encodes Rep protein (rep gene), and the remainder half encodes a capsid protein (cap gene). At least thirteen serotypes (AAV 1-13) have been known to date as wild-type AAVs which are infectious to human.
Typical recombinant adeno-associated viral vectors (rAAV vectors) have a genome structure in which rep gene and cap gene of the AAV genome are replaced with desired genes and the like. One example of the method for preparing an rAAV vector includes a method including introducing into host cells such as 293 cells collectively a vector plasmid in which an intended gene is inserted between ITRs at both terminals of the AAV, a helper plasmid for supplying a viral protein in need of replication of the AAV or formation of viral particles, and an adenovirus helper plasmid having a part of gene region serving as a helper action of the adenovirus in need of proliferation of the AAV, to produce an rAAV vector in the nucleus of the host cells. When the rAAV vector is used for gene transduction of the cells, it is necessary to prepare an rAAV vector having a sufficiently high titer. Therefore, the determination of the titer is essential in the preparation of the rAAV vector.
As the determination method for titer of rAAV, southern blotting method, dot blotting method, and real-time PCR method or the like has been utilized. Among them, it is said that it is preferable to utilize the real-time PCR method, from the viewpoint of convenience and rapidness of the operation, or the like. The ITR of the rAAV vector has a secondary structure which is difficult to perform PCR amplification. Therefore, as a target sequence for the determination according to real-time PCR of the titer of the rAAV vector, a foreign gene inserted into the vector has been utilized. However, when a foreign gene is utilized as the target sequence, it is necessary to design primers utilized in PCR for every vector constructed. Recently, a report has been made on a determination method for titer of an rAAV vector according to real-time PCR using a TaqMan probe, in which the ITR sequence alone is a target sequence (Non-Patent Publication 1). However, it is necessary to synthesize a double labeled nucleic acid probe in the real-time PCR using the TaqMan probe, so that it is more costly as compared to quantitative real-time PCR using an intercalating dye.